Mapping discontinuous epitopes recognized by human enhancing antibodies In the last decade, mass spectrometry has been employed by more and more researchers for identifying the proteins in a macromolecular complex as well as for defining the surfaces of their binding interfaces. This characterization of protein:protein interfaces usually involves at least one of several different methodologies in addition to the actual mass spectrometry. For example, limited proteolysis is often used as a first step in defining regions of a protein that are protected from proteolysis when the protein of interest is part of a macromolecular complex. Other techniques used in conjunction with mass spectrometry for determining regions of a protein involved in proteinprotein interactions include chemical modification, such as covalent cross-linking, acetylation of lysines, hydrogen-deuterium exchange, or other forms of modification. We initiated a project in which we undertook experiments to 1) probe an epitope on membrane embedded HIV gp160 recognized by the human neutralizing Ab 4E10 and probed the discontinuous epitope on the HIV gp120 trimeric spike recognized by a human neutralizing Ab. In these experiments we used a stable gp160 trimeric spike engineered using LipoParticles. LipoParticles are a patented process to express integral membrane proteins on a non-infectious particle. MAb 2909 recognizes a discontinuous epitope on the native trimeric spike conformation of the HIV envelope protein. It does not recognize monomeric gp120 or gp160. We attempted several approaches to isolating amounts of expressed membrane-protein sufficient for mass spectral analyses. These methods used techniques including several centrifugation-based approaches and peptide shaving. None of these methods yielded sufficient material for MS-based analysis. We also attempted these methods on the model CXCR4 protein overexpressed on LipoParticles. Although the levels of this protein were significantly higher than the level of expressed gp160, the level was too low for MS-detection, both as determined experimentally and computationally. Hydrogen/Deuterium exchange studies have been initiated to further characterize biologically important structural changes undergone by HIV reverse transcriptase. This project is in collaboration with London/Mueller (LSB). We also began to explore improved methods for identifying site-specific glycosylation patterns on proteins with the goal of using reverse phase-reverse phase 2-Dimensional- Ultra Performance (high pressure) liquid chromatographic separation combined with MS/MS. In this procedure, 5 distinct fractions are eluted (at different pHs) from the 1st column with each fraction undergoing further separation on the second reverse phase column. This enhances glycopeptide sensitvity by shifting the elution of glycopeptides to fractions with significantly less abundant peptides.